Part:BBa_K3806016:Design

Theophylline-binding aptazyme regulating lacZ expression (semi-cRBS). With T7 promoter.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3204
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2321
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This biobrick represents the DNA template for the expression of the lacZ reporter gene in a T7 based cell-free expression system. Expression is regulated by a theophylline binding aptazyme [1]. The dsRNA sequence was PCR amplified from BBa_K3806014. The forward primer used allowed for the change of the C and T (U in the aptazyme) nucleotides in the antisense strand into G and A, respectively, resulting in a partial mismatch to the RBS (Fig. 1).

Fwd primer: 5’ AATTTAATACGACTCACTATAGGGAAACAAACAAAGACCTTGCTGTCACCGGAATG 3’

Rv primer: 5’ CACACAGGAAACAGCTATGACCATG 3’

Fig. 1 Secondary structure of the transcribed products from (A) BBa_K3806014 (cRBS) (B) BBa_K3806016 (semi-cRBS). The RBS is shaded in magenta and the two modified nucleotides of the antisense strand are pointed with a black arrow.

For more information in this design, visit the Design page of the TU Delft 2021 team.

References

1. [1] Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.